1. Field of the Invention
The present invention is directed to methods for dissociating Fc containing molecules from complexes of Protein A/Fc containing molecules in mixtures.
2. Related Background Art
Purification of target proteins is often encumbered by poor DNA removal due to DNA/protein interactions. DNA/protein interactions are more problematic in the purification of highly anionic target proteins, e.g., sulfated proteins. Purification of proteins, e.g., proteins containing immunoglobulin domains, is also often difficult based on the low pH of dissociation required to separate Fc containing molecules from Protein A. The identification of methods useful in removing DNA from target proteins and the identification of methods for removing Protein A from target proteins would be of great benefit in the purification of various proteins.
International Publication No. WO 01/72769 describes methods for isolating and purifying highly anionic target proteins and target proteins comprising immunoglobulin domains, for example, sulfated proteins. Anionic proteins are proteins which have a net negative charge. Sulfated proteins are proteins in which the net negative charge is due to at least about one (1) sulfated residue. Sulfation of a target protein refers to the substitution of at least one hydroxyl group (—OH) with —SO4H on or between amino acid(s) contained within the target protein. In a preferred embodiment, the sulfated protein has at least about one (1) sulfate group. Sulfated proteins containing at least about two (2), three (3), four (4), five (5), six (6) or more sulfate groups are also encompassed by the present methods, e.g., six sulfate groups on the N-terminal tyrosines as embodied in PSGL-1 (P-Selectin Glycoprotein Ligand-1).
In particular, International Publication No. WO 01/72769 discloses, in one aspect, a method for purifying highly anionic target proteins comprising the steps of ion exchange chromatography under appropriate conditions for the purification of the target proteins. As an example, the disclosed method provides for (1) contacting the sample with a substrate capable of reversibly binding charged molecules whereby the target proteins bind to the substrate, (2) washing the substrate with a first wash solution under appropriate conditions whereby a plurality of proteinaceous and non-proteinaceous impurities in the sample either do not bind or are washed off the substrate while the highly anionic target proteins remain bound, (3) eluting the sample with a first elution solution wherein the first elution solution comprises a salt solution at a high molar concentration, and (4) collecting the eluted sample containing the purified anionic target proteins.
In one embodiment, it is disclosed that the pH of the first wash solution is about 4.0 to 8.0. In another embodiment, it is disclosed that the pH of the first wash solution is about 6.5.
In a preferred embodiment, it is disclosed that the highly anionic target protein is a sulfated protein and the impurities include a sulfated form of the target protein.
International Publication No. WO 01/72769 also discloses that the eluted sample from the ion exchange chromatography purification which contains the purified target proteins can be further purified. This further purification, for example, comprises the steps of hydrophobic interaction and/or metal chelate chromatography under appropriate conditions for the purification of the highly anionic target proteins. For example, this further purification provides for the steps of (1) passing the eluted sample containing the target proteins through a metal chelate chromatography column or a hydrophobic interaction chromatography column whereby the eluted sample is captured on the column, (2) washing the column with a second wash solution under appropriate conditions whereby DNA/histone complexes contained in the sample are dissociated, (3) eluting the sample with a second elution solution, and (4) collecting the eluted sample containing the purified highly anionic target proteins.
In one disclosed embodiment, the second wash solution comprises a high salt concentration and the second elution solution comprises a lower salt concentration than the second wash solution. For example, under hydrophobic interaction chromatographic conditions, the concentration of the salt in the second wash solution is about 4M, and the concentration of the salt in the second elution solution is about 0.48M. Alternatively, under hydrophobic interaction chromatography the second wash solution is selected from the group consisting of (a) a solution comprising NaCl at about 4M and Tris at about 20 mM and a pH of about 7.4, (b) a solution comprising isopropanol at about 5% and ammonium sulfate at about 1.2M, (c) a solution of ethanol at about 5% and ammonium sulfate at about 1.2M, and (d) a solution of ethanol of about 5% and NaCl at about 4M.
It is further disclosed that under iron chelation chromatographic conditions, for example, the second wash solution comprises a salt concentration of about 2M, and the second elution solution comprises a salt concentration of about 200 mM to 1M. Alternatively, under iron chelation chromatographic conditions, the second wash solution comprises MES at about 40 mM, NaCl at about 2M, and imidazole at about 5 mM, and the second elution solution comprises a solution of MES at about 40 mM, NaCl at about 1M, and imidazole at about 35 mM.
International Publication No. WO 01/72769 discloses that the target proteins have at least about one (1) sulfation(s). Anionic target proteins having at least about two (2), three (3), four (4), five (5), six (6), or more sulfations are also disclosed, e.g., PSGL-1 proteins. Anionic proteins capable of being purified by the disclosed methods can be naturally occurring or recombinant proteins.
Also disclosed in International Publication No. WO 01/72769 is a method for the purification of highly anionic proteins comprising an immunoglobulin domain (e.g., an immunoglobulin Fc domain), for example, a PSGL-Ig fusion protein. This disclosed method comprises the steps of (1) contacting the sample with a substrate capable of binding the Fc portion of the target protein comprising an immunoglobulin domain whereby the target molecules bind to the substrate, (2) washing the substrate with a first wash solution under appropriate conditions to wash away contaminants contained in the sample, (3) eluting the sample with a first elution solution wherein the pH of the first elution solution is low, e.g., about 4.0, preferably about 3.7, and (4) collecting the eluted sample containing the purified anionic target proteins.
It is further disclosed that the eluted sample from the Fc binding substrate which contains the purified highly anionic target proteins comprising an immunoglobulin domain may be further purified. For example, further purification comprises the steps of (1) contacting the eluted sample containing the purified anionic target proteins comprising an immunoglobulin domain with a substrate capable of reversibly binding charged molecules whereby a plurality of proteinaceous and non-proteinaceous impurities in the sample either do not bind or are washed off the substrate while the target proteins remain bound to the substrate, (2) washing the substrate with a second wash solution wherein the pH of the second wash solution is low, e.g., about 4.0, preferably about 3.8, (3) eluting the sample with a second elution solution, and (4) collecting the eluted sample containing the purified anionic target proteins comprising an immunoglobulin domain.
In one aspect, it is disclosed that the target proteins comprising an immunoglobulin domain have at least about one (1) sulfation(s). Immunoglobulins comprising proteins with at least two (2), three (3), four (4), five (5), six (6), or more sulfations are also disclosed by International Publication No. WO 01/2769, e.g., PSGL-Ig.
In a preferred embodiment, the purification methods disclosed provide purified highly anionic target proteins and purified highly anionic proteins comprising an immunoglobulin domain (e.g., PSGL-Ig) at least about 99.9% pure of contaminating proteins.
In another disclosed embodiment, the purification methods of the invention removes at least about 95% or 2.5 log10 removal value (LRV) of the contaminating DNA from the highly anionic target proteins and the highly anionic proteins comprising an immunoglobulin domain.
A method, however, for dissociating Fc containing molecules from complexes of Protein A/Fc containing molecules in mixtures would be highly desirable.
This application is related to prior-filed provisional patent application No. 60/193,351, filed on Mar. 27, 2000, prior-filed U.S. patent application Ser. No. 09/819,157, filed on Mar. 27, 2001 (now U.S. Pat. No. 6,933,370), and prior-filed international application No. PCT/US01/09815, filed Mar. 27, 2001. The entire contents of each of the above-referenced applications are incorporated herein by this reference.